Hybridization-sensitive fluorescent DNA probe with self-avoidance ability.

Hybridization-sensitive fluorescent probes have an inherent disadvantage: self-dimerization of the probe prevents the fluorescence quenching prior to hybridization with the target, resulting in a high background signal. To avoid self-dimerization of probes, we focused on a base pair formed by 2'-deoxyinosine (I) and N(4)-ethyl-2'-deoxycytidine (E). I and E bases form more stable base pairs with cytosine and guanine, respectively, compared with an I/E base pair. New hybridization-sensitive fluorescent probes, IE probes, were prepared containing three unnatural nucleotides, I, E and D(514) as a doubly thiazole orange-labeled nucleotide. The IE probes had low thermostability, sufficient to avoid self-dimerization. Absorption spectra of the IE probes exhibited a hybridization-dependent shift of the absorption maximum, suggesting that excitonic interaction was working between the thiazole orange dyes in the probe. Interdye excitonic interaction of IE probes was very effective; thus, replacement of guanine and cytosine with I and E improved the ratio of fluorescence intensities after and before hybridization (I(hybrid)/I(nonhybrid)). Although a significant weakness in fluorescence intensity was observed for several IE probes after hybridization with the target sequence when both or either of the bases adjacent to D(514) is E, a dramatic recovery of the fluorescence intensity of hybrids was observed when any E adjacent to D(514) was replaced with cytosine. Improvement of the I(hybrid)/I(nonhybrid) value by incorporation of I and E helped the design of a long probe sequence for mRNA imaging.